Protease has long been used in industry, and has found utility in a diversity of fields, including detergents such as laundry detergents, fiber modifying agents, leather processing agents, cosmetic compositions, bath additives, food-modifying agents, and pharmaceuticals. Of these, proteases for detergent use are produced in the largest amounts on an industrial scale. Examples of such known proteases that are derived from Bacillus include ALCALASE, SAVINASE (registered trademarks; Novozymes), MAXACAL (registered trademark; Genencor), BLAP (registered trademark; Henkel), and KAP (Kao Corporation).
The purpose of incorporating protease into a detergent is to degrade protein soil adhering to clothes. Such soil actually is a “complex” soil formed of a plurality of organic and inorganic components, including not only proteins but also lipids originating from sebum, solid particles, and other substances. Therefore, demand has arisen for a detergent having excellent detergency to such complex soil.
Under the above situation, some of the present inventors had previously discovered several species of alkaline protease which have a molecular weight of about 43,000, exhibit a sufficient casein-degrading activity even in the presence of a fatty acid at a high concentration, and also exhibit excellent detergency not only to proteins but also to complex soils which include sebum and other substances, and filed a patent application therefor (see Patent Publication WO99/18218). Since the discovered alkaline proteases differ from subtilisin (which is a conventionally known serine protease derived from a microorganism belonging to the genus Bacillus) in terms of molecular weight, primary structure, enzymological characteristics and resistance to oxidants (the alkaline proteases are strongly resistant to oxidants) their classification into a new subtilisin subfamily has been proposed (see, for example, Saeki et al., Biochem. Biophys. Res. Commun., 279, 313-319, 2000).
In order to industrially mass-produce protease having excellent detergency, productivity thereof must be enhanced. To this end, a variety of methods are envisaged, including selective mutant breeding of enzyme-producing bacteria, and alteration of a gene coding for protease or a gene related to regulation of expression thereof so as to increase the amount of secreted protein. Alternatively, the gene coding for protease is modified so that an enhanced specific activity is obtained. From these viewpoints, the present inventors have previously discovered mutated alkaline protease exhibiting improved protein secretion ability and enhanced specific activity (Japanese Patent Application Nos. 14-304230, 14-304231, and 14-304232 Application Laid-Open (kokai) Nos. 2004-000122 and 2004-057195).
However, further improvement in productivity is desired for producing the enzyme on a large scale at low cost. To answer this, means for improving the amount of secreted protein and specific activity has become of keen interest.